Drug resistance is influenced by molecular mechanisms that affect intracellular drug exposure and a drug’s ability to reach its protein target. Visualizing subcellular behavior of small-molecule drugs requires single-cell chemical analysis with high spatial and temporal resolution. In this work, we utilized label-free hyperspectral stimulated Raman scattering (hsSRS) microscopy to directly visualize and quantify tyrosine kinase inhibitor (TKI) drugs inside living cells. We report our live cell imaging acquisition and processing pipeline and demonstrate the capability of this hsSRS imaging method to characterize the roles of organic cation transporters (OCTs) on drug uptake. We observe significant transporter-dependent changes in intracellular drug concentration of multiple TKIs in response to the inhibition of OCTs.
SignificanceThe molecular mechanisms driving the progression from nonalcoholic fatty liver (NAFL) to fibrosing steatohepatitis (NASH) are insufficiently understood. Techniques enabling the characterization of different lipid species with both chemical and spatial information can provide valuable insights into their contributions to the disease progression.AimWe extend the utility of stimulated Raman scattering (SRS) microscopy to characterize and quantify lipid species in liver tissue sections from patients with NAFL and NASH.ApproachWe applied a dual-band hyperspectral SRS microscopy system for imaging tissue sections in both the C–H stretching and fingerprint regions. The same sections were imaged with polarization microscopy for detecting birefringent liquid crystals in the tissues.ResultsOur imaging and analysis pipeline provides accurate classification and quantification of free cholesterol, saturated cholesteryl esters (CEs), unsaturated CE, and triglycerides in liver tissue sections. The subcellular resolution enables investigations of the heterogeneous distribution of saturated CE, which has been under-examined in previous studies. We also discovered that the birefringent crystals, previously found to be associated with NASH development, are predominantly composed of saturated CE.ConclusionsOur method allows for a detailed characterization of lipid composition in human liver tissues and enables further investigation into the potential mechanism of NASH progression.
Nonalcoholic fatty liver disease (NAFLD) is characterized by the accumulation of lipid in the hepatocytes (steatosis), which can progress to fibrosing steatohepatitis (NASH). The molecular mechanisms driving this progression are insufficiently understood. A leading hypothesis is lipotoxicity, which postulates that specific lipid species can trigger the cascade of inflammation leading to liver damage. In this study, we used label-free stimulated Raman scattering (SRS) microscopy to characterize the distribution of free cholesterol, cholesteryl esters, and triglycerides of different saturation levels in liver tissue from patients with histologically diagnosed NAFLD versus NASH. By probing the intrinsic vibrational frequencies of lipid molecules in the C–H and the fingerprint regions, we can localize and classify different lipid molecules in lipid droplets via spectral unmixing. We report our developed image acquisition and processing pipeline in this paper and demonstrate example applications such as examining the composition of previously described cholesterol crystals. We discovered that most of the birefringent liquid crystals presumed to be free cholesterol crystals in NASH tissues are predominantly composed of saturated cholesteryl esters. Our method provides a detailed characterization of the lipid composition in NAFLD tissues and allows us to probe further into the potential mechanism of NASH development.
Stimulated Raman scattering (SRS) microscopy is a powerful chemical imaging tool for visualizing biomolecule distribution in biological cells and tissues. Recent research has focused on SRS imaging in the C-H region due to the strong signals from lipids and proteins. However, these signals are regarded as non-specific. To improve the specificity of C-H imaging, we sought to use advanced machine learning to extract hidden information from C-H SRS imaging. This is possible because cells in tissue often have distinct sizes, shapes, and compositions. In this talk, I will present our recent efforts on machine learning/deep learning augmented cell imaging and classification with SRS. This approach potentially enables label-free mapping and tracking of different cells in various tissue.
Particles in biopharmaceutical products present high risks due to their detrimental impacts on product quality and safety. Quantification and identification of particles in drug products are important to understand particle formation mechanisms, which could help control strategy development on particle formation during formulation development and manufacturing process. However, existing analytical techniques such as MFI and HIAC lack the sensitivity and resolution to detect particles with sizes smaller than 2 μm. More importantly, there is no chemical information available for determining particle content. In this work, we develop a Stimulated Raman Scattering (SRS) microscopy technique that overcomes these challenges by monitoring the C-H Raman stretching modes of the proteinaceous particles and a common contaminant (silicone oil). By comparing the relative signal intensity and spectral features of each component, most particles can be classified as protein, protein-silicone oil, or silicone oil. Our method has the capacity to quantify aggregation in protein therapeutics with chemical and spatial information in a label-free manner, potentially allowing high throughput screening or investigation of aggregation mechanisms.
T cell differentiation has warranted intense study to understand the mechanisms behind the adaptive immune system. While much of the research so far has relied on antibody staining and flow cytometry separation to isolate and study T cells, we present hyperspectral stimulated Raman scattering (SRS) microscopy as a potential label-free imaging method to directly observe and characterize T cells. We show that a deep learning model can be trained to identify and classify T cell differentiations from hyperspectral SRS images with 99% accuracy. We also show that fluorescent T cells in lymph node tissue can be predicted from SRS images, demonstrating potential towards an entirely label-free in-situ imaging strategy. SRS microscopy augmented with deep learning shows strong promise towards label-free in situ observation of T Cells.
In recent studies, stimulated Raman scattering (SRS) and transient absorption microscopy (TAM) have been employed for label-free mapping of biomolecules (e.g., proteins and lipids) in brain tissues and hemoglobin in red blood cells, respectively. In this study, we combined SRS and TAM to simultaneously image cell densities and capillary structure in vivo at the highest reported imaging depth, ~300 µm, for either technique. This multimodal approach resulted in label-free identification of endothelial cells and pericytes in vivo with 90% accuracy using a machine learning classifier. Simultaneous two-photon excited fluorescence microscopy serving as the ground truth.
Hyperspectral stimulated Raman scattering (hsSRS) microscopy provides rich chemical and spatial information not regularly available to traditional microscopy methods. However, analysis of hsSRS images is often confounded by convolved and overlapping spectral features requiring use of machine learning methods to extract information. Here, we demonstrate the use of our recently published deep learning architecture (the U-within-U-Net) designed for hyperspectral images on hsSRS images. We demonstrate segmentation, classification, and prediction of orthogonal imaging modalities. We also show the architecture is applicable to other hyperspectral imaging modalities with implications for remote sensing and mass spectrometry imaging.
The brain is an enormously complex organ that consumes a substantial amount of body energy. Understanding how brain function requires the ability to not only map out the cellular structure but also probe functional processes. Multiphoton fluorescence microscopy has played a crucial role in current investigations due to the wide variety of synthetic dyes and proteins available for imaging brain cells and neuronal activities at high spatial and temporal resolution. However, it has limited capability to resolve multiple features simultaneously. We combine multiphoton fluorescence with label-free nonlinear imaging techniques including transient absorption microscopy and stimulated Raman scattering microscopy to expand the structural and functional features that can be imaged simultaneously. With this platform, we demonstrate the reconstruction of axonal features and microvascular networks together with the mapping of cellular organization.
Chemical imaging of living cells is critically important for understanding the function and pathophysiology of biological systems such as the brain. The main obstacles are the limited amount of analyte in a single cell and the need for noninvasive in situ analysis in order to preserve cell function and microenvironmental information. We apply label-free chemical imaging methods to quantify the spatiotemporal distribution of important biomolecules at subcellular resolution. In combination with deep learning algorithms, we aim to build an integrated chemical imaging platform to study a wide range of normal and diseases processes in the brain.
Solid dosage formulations remain the most important dosage forms for pharmaceuticals. In all solid formulations, the most important properties are the stability and bioavailability of the active pharmaceutical ingredient (API). Different polymorphs of API crystals often differ in the physicochemical properties like chemical and physical stability, solubility and dissolution. These differences leads to variability in drug efficacy, bioavailability, and even toxicity. A thorough understanding of how excipients and physical processing impact the polymorphism, stability, and dissolution rate of API is crucial to accelerate drug development and regulatory approval. However, currently no technology can monitor the dynamic chemical changes of solid formulation in situ at sub micrometer resolution during processing or dissolution.
SRS microscopy is a powerful chemical imaging technique that can potentially address these challenges. In particular, it allows label-free imaging of APIs and excipients at high spatial and temporal resolution. Here we present our recent work on using hyperspectral SRS microscopy to resolve polymorphic changes of APIs at low drug loading. In addition, we demonstrate that we can monitor the dissolution of individual entecavir drug crystals in a slow-release implant drug sample. Together, these experiments demonstrate that hyperspectral SRS microscopy can be a valuable technique for resolving details of drug instability and drug dissolution in formulation research.
Improving the sensitivity of mammograms in breast cancer screening has increased the detection of suspicious findings such as calcifications and masses at the cost of a high false positive rate (55-85%). Additionally, the diagnostic interpretation of biopsies varies (75.3% concordance), leading to suboptimal treatments and poor patient outcomes. The goal of this pilot study is to investigate whether the chemical composition of breast calcifications, present in more than 80% of mammograms, can be used to improve breast lesion classification. We hypothesized that the spatial and compositional variation of breast calcifications strongly correlates with breast malignancy. To test this hypothesis, we used an advanced Raman imaging technique called hyperspectral stimulated Raman scattering (hsSRS) microscopy to study 12 patient cases (30 calcifications). We characterized unique Raman signatures of type I (calcium oxalate) and type II (calcium hydroxyapatite) calcifications in archival breast tissue at high speed and spatial resolution with hsSRS microscopy. We found that the carbonation level of hydroxyapatite decreases when comparing benign and atypical ductal hyperplasia. However, the average carbonation of hydroxyapatite was highly variable in fibroadenoma cases (3±0.6%) and DCIS (4±1.1%). In the case of DCIS, the carbonation of hydroxyapatite varied relative to the grade and the neoplastic microenvironment (nearby inflammation, necrosis, and more.) In high-grade DCIS, the carbonation was lowest around the periphery where the contact with neoplastic cells was present. Our preliminary results indicate that microcalcifications change with the neoplastic microenvironment. Further studies of neoplastic progression in association with microcalcifications can improve the statistical value of the correlation.
Hyperspectral Stimulated Raman scattering (hsSRS) microscopy has recently emerged as a powerful non-destructive technique for label-free chemical imaging of biological samples. In most hsSRS imaging experiments, the SRS spectral range is limited by the total bandwidth of the excitation laser to ~300 cm-1 and spectral resolution of ~25 cm-1. Here we present a novel approach for broadband hsSRS microscopy based on parabolic fiber amplification to provide linearly chirped broadened Stokes pulses. This novel hsSRS instrument provides >600 cm-1 spectral coverage and ~10 cm-1 spectral resolution. We further demonstrated broadband hsSRS imaging of the entire Raman fingerprint region for resolving distribution of major biomolecules in fixed cells. Moreover, we applied broadband hsSRS in imaging amyloid plaques in human brain tissue with Alzheimer’s disease.
We demonstrate a fiber-based two-color source of picosecond pulses for coherent Raman scattering (CRS) microscopy.
An Yb-doped fiber laser combined with a divided-pulse amplifier produce up to 3 W of power tunable from 1030 nm to
1040 nm. A normal dispersion photonic crystal fiber is used to blue-shift the pulses through seeded four-wave mixing.
Pulses with up to 150 mW of average power are produced, tunable between 770 nm and 800 nm. Imaging of animal
tissue and cells is demonstrated.
Multiple scattering is a significant obstacle in the optical imaging of biological samples. However, it is possible to
reverse its effects through optical phase conjugation (OPC) of the scattered field. We perform digital OPC (DOPC)
utilizing a spatial light modulator (SLM) and a Sagnac interferometer geometry. This design permits a simple and robust
DOPC implementation, which we demonstrate experimentally. We exploit the beam-shaping flexibility of the SLM to
demonstrate the possibility to enhance either the optical power transmission or the light focusing ability of the DOPC
process.
Wide-field reflection phase microscopy is highly desired for depth-resolved measurement of cellular structures without
the need for raster scanning. We report a low coherence reflection phase microscope based on time-domain optical
coherence tomography and off-axis interferometry. The setup uniquely provides the desired angular shift to the reference
beam for off-axis interferometry while promising equal path length across the whole reference beam. We show sub-nanometer
path-length sensitivity of our instrument and demonstrate high-speed imaging of membrane fluctuations in
eukaryotic cells.
We present the light scattering of individual Plasmodium falciparum-parasitized human red blood cells (Pf-RBCs), and demonstrate progressive alterations to the scattering signal arising from the development of malaria-inducing parasites. By selectively imaging the electric fields using quantitative phase microscopy and a Fourier transform light scattering technique, we calculate the light scattering maps of individual Pf-RBCs. We show that the onset and progression of pathological states of the Pf-RBCs can be clearly identified by the static scattering maps. Progressive changes to the biophysical properties of the Pf-RBC membrane are captured from dynamic light scattering.
Nonlinear microscopies (most commonly, two-photon fluorescence, second harmonic generation, and coherent
anti-Stokes Raman scattering (CARS)) have had notable successes in imaging a variety of endogenous and exogenous targets
in recent years. These methods generate light at a color different from any of the exciting laser pulses, which makes the
signal relatively easy to detect. Our work has focused on developing microscopy techniques using a wider range of
nonlinear signatures (two-photon absorption of nonfluorescent species, self phase modulation) which have some specific
advantages - for example, in recent papers we have shown that we can differentiate between different types of melanin
in pigmented lesions, image hemoglobin and its oxygenation, and measure neuronal activity. In general, these signatures
do not generate light at a different color and we rely on the advantages of femtosecond laser pulse shaping methods to
amplify the signals and make them visible (for example, using heterodyne detection of the induced signal with one of the
co-propagating laser pulses). Here we extend this work to stimulated Raman and CARS geometries. In the simplest
experiments, both colors arise from filtering a single fs laser pulse, then modulating afterwards; in other cases, we
demonstrate that spectral reshaping can retain high frequency resolution in Raman and CARS geometries with
femtosecond laser pulses.
As some of the most ubiquitous and biologically important natural pigments, melanins play essential roles in the photoprotection of skin. Changes in melanin production could potentially be useful for clinical diagnosis of the progression stage of melanoma. Previously we demonstrated a new method for imaging melanin distribution in tissue with two-color transient absorption microscopy. Here we extend this study to longer wavelengths and show that we are able to image melanin in fixed thin skin slices with higher signal-to-noise ratios (SNRs) and demonstrate epimode imaging. We show that both photothermal effects and long-lived excited states can contribute to the long-lived signal. Eumelanin and pheomelanin exhibit markedly different long-lived excited state absorption. This difference should enable us to map out their respective distribution in tissue samples with subcellular resolution. This technique could provide valuable information in diagnosing the malignant transformation of melanocytes.
The ability to perform high-resolution imaging of microvasculature and its oxygenation is very important in studying early tumor development. Toward this goal, we improved upon our excited state absorption (ESA)–based imaging technique to allow us to not only image hemoglobin directly but also differentiate between oxy- and deoxyhemoglobin in tissue. We demonstrate the separation of arterioles from venules in a live nude mouse ear using our imaging technique.
We have demonstrated a new optical microscopy technique for imaging microvasculature without any labeling. With a
very sensitive two-color excited state absorption (ESA) measurement method, we demonstrated that oxy-hemoglobin
and deoxy-hemoglobin show distinct excited state dynamics. Since this is a collinear measurement, we can readily apply
it to the microscopic study of biological tissue. We have already demonstrated in vivo imaging of blood vessels in the
nude mouse ear. Here we optimized the excitation and detection pulse train toward longer wavelengths, where tissue
scatters less and greater penetration depth can be obtained. More importantly, we are able to separate arterioles from
venules by employing different pump and probe wavelength combinations. This provides a powerful method to image
blood vessels and their oxygenation level at the same time with micrometer resolution.
As a main pigment in skin tissues, melanin plays an important role in photo-protecting skin from UV radiation. However, melanogenesis may be altered due to disease or environmental factors; for example, sun exposure may cause damage and mutation of melanocytes and induce melanoma. Imaging pigmentation changes may provide invaluable information to catch the malignant transformation in its early stage and in turn improve the prognosis of patients. We have demonstrated previously that transmission mode, two-photon, one- or two-color absorption microscopy could provide remarkable contrast in imaging melanin in skin. In this report we demonstrate significantly
improved sensitivity, so that we are now able to image in epi-mode (or back reflection) in two-photon absorption. This
improvement makes possible for us to characterize the different types of pigmentation on the skin in vivo at virtually any location. Another finding is that we can also image transient photothermal dynamics due to the light absorption of melanin. By carefully choosing excitation and probe wavelengths, we might be able to image melanin in different structures under different micro-environments in skin, which could provide useful photochemical and photophysical insights in understanding how pigments are involved in photoprotection and photodamage of cells.
We develop a new approach in imaging nonfluorescent species with two-color two-photon and excited state absorption microscopy. If one of two synchronized mode-locked pulse trains at different colors is intensity modulated, the modulation transfers to the other pulse train when nonlinear absorption takes places in the medium. We can easily measure 10−6 absorption changes caused by either two-photon absorption or excited-state absorption with a RF lock-in amplifier. Sepia melanin is studied in detail as a model system. Spectroscopy studies on the instantaneous two-photon absorption (TPA) and the relatively long-lived excited-state absorption (ESA) of melanin are carried out in solution, and imaging capability is demonstrated in B16 cells. It is found that sepia melanin exhibits two distinct excited states with different lifetimes (one at 3 ps, one lasting hundreds of nanoseconds) when pumped at 775 nm. Its characteristic TPA/ESA enables us to image its distribution in cell samples with high resolution comparable to two-photon fluorescence microscopy (TPFM). This new technique could potentially provide valuable information in diagnosing melanoma.
We have demonstrated a new method for imaging melanin with two-color excited state absorption microscopy. If one of
two synchronized mode-locked pulse trains at different colors is intensity modulated, the modulation transfers to the
other pulse train when nonlinear absorption takes place in the medium. We can easily measure 10-6 absorption changes
caused by either instantaneous two-photon absorption or relatively long lived excited state absorption with a RF lock-in
amplifier. Eumelanin and pheomelanin exhibit similar excited state dynamics. However, their difference in excited state
absorption and ground state absorption leads to change in the phase of the transient absorption signal. Scanning
microscopic imaging is performed with B16 cells, melanoma tissue to demonstrate the 3D high resolution imaging
capability. Different melanosome samples are also imaged to illustrate the differences between eumelanin and
pheomelanin signals. These differences could enable us to image their respective distribution in tissue samples and
provide us with valuable information in diagnosing malignant transformation of melanocytes.
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